Method of determining cytokine dosage for myelosuppressive state

ABSTRACT

The invention provides kits and methods for evaluating the myelosuppressive state of a patient. These methods and kits provide a useful adjunct for cytotoxic and myelosuppressive therapies. By establishing threshold levels of certain cytokines as a surrogate for myelosuppression, treatment protocols can be optimized to reduce myelotoxicity, while maximizing effective dose. Measured levels of one or more cytokines in a patient subjected to cytotoxic therapy, relative to a normal population, may be used to determine the dose of a hematopoietic cytokine to be administered to the patient.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/173,550 (allowed), filed on Jun. 14, 2002, which was acontinuation-in-part of U.S. patent application Ser. No. 09/482,730 (nowissued U.S. Pat. No. 6,649,352), filed Jan. 14, 2000, which claimed thebenefit of Provisional Application No. 60/118,071, filed Jan. 29, 1999,each hereby incorporated by reference in its entirety.

GOVERNMENT FUNDED RESEARCH

This work was supported in part by United States Public Health Servicegrant RO1 CA49995 (RDB) and R23 CA39841 (DMG) from the NationalInstitutes of Health. The U.S. Government may have certain rights in theinvention.

FIELD OF THE INVENTION

The present invention concerns methods and compositions for amelioratingthe effects of therapy with myelosuppressive agents. In particularembodiments, the methods and compositions may allow determining the doseof a hematopoietic agent to be administered to a patient before or aftermyelosuppressive therapy.

BACKGROUND OF THE INVENTION

Both chemotherapy and radioimmunotherapy induce dose-limitingmyelosuppression. In fact, chemotherapy-induced myelosuppression is themost common dose-limiting, and potentially fatal, complication of cancertreatment. Maxwell et al., Semin. Oncol. Nurs. 8:113-123 (1992);Blijham, Anticancer Drugs 4:527-533 (1993). Drug-induced hematopoietictoxicity is a common reason for curtailing high dose chemotherapy incancer patients (Boesen et al., Biotherapy 6:291-302 (1993)), and higherdose chemotherapy is only possible in conjunction with bone marrowtransplantation (BMT), autologous stem cell infusion, and treatment withhematopoietic growth factors.

During the recovery period after anticancer myelosuppressive therapy,hematopoietic progenitor cells become mitotically active in order toreplenish the marrow compartment and remain hyperproliferative evenafter normalization of peripheral white blood cells (pWBCs) andplatelets (PLTs). At this stage, the progenitors are more radio- andchemo-sensitive. Dosing patients with additional cytotoxic therapyduring this phase will likely result in more severe toxicity.

As a general model of myelosuppressive therapy, acute damage andrecovery of hematopoietic stem and precursor cells following whole-bodyirradiation also has been studied extensively. Testa et al., AnticancerRes. 5:101-110 (1985); Sado et al., Int. J. Radiat Biol 53:177-187(1988); Meijne et al., Exp. Hematol. 19:617-623 (1991). External beamirradiation results in long-term damage of hematopoietic stem cells,which manifests with the presence, but at sub-optimal levels, ofmitotically active, hematopoietic progenitor cells (CFU-S) 3-6 monthsafter treatment. Lorimore et al., Int. J. Radiat Biol 57:385-393 (1990);Lord et al., Int. J. Radat. Biol. 59:211-218 (1991). Persistentdepletion of femoral and splenic CFU-S (colony forming unit-spleen),CFU-GM (colony forming unit-granulocytic-monocytic) and BFU-E (burstforming unit-erythroid) can occur, even though the peripheral bloodcontains normal cell numbers. Grande et al., Int. J. Radiat. Biol.59:59-67 (1993). Severe reduction in the supportive stroma has also beenreported. Tavassoli et al., Exp. Hematol. 10:435-443 (1992). Followingradiation exposure, recovery proceeds by repair of sublethal cellularinjury and compensatory cellular repopulation by the surviving fraction.Hall in RADIOBIOLOGY FOR THE RADOBIOLOGIST (Harper & Row 1978); Jones etal., Radiation Res. 128:256-266 (1991).

Normal white blood cell (WBC; >4000/mm³) and platelet (PLT;>100,000/mm³) counts are the usual markers for patient tolerance torepetitive myelosuppressive treatment. However, preclinical and clinicalevidence suggests that peripheral counts are not a reliable surrogatefor predicting complete myelosuppressive recovery. Although WBC and PLTcounts may appear normal, the primitive stem and progenitor cellcompartments are not fully recovered from previous myelosuppressivetherapy.

Further cytotoxic treatment while stem cells and progenitor cells arerapidly proliferating can result in more severe myelotoxicity or evendeath. One solution to this problem is to collect bone marrow (BM)aspirates and use a long-term culture system to quantitate highproliferative potential CFC(HPP-CFC) or long term culture initiatingcells (LTC-IC). Eaves et al, Tiss. Culture Meth. 13:55-62 (1991);McNiece et al., Blood 75:609-612 (1989). While this method can providethe needed information, such assays take 3-6 weeks to perform, and thusare not clinically useful.

During hematopoiesis, pluripotent stem cells differentiate andproliferate in multiple lineages. The process proceeds under thepermissive influence of “early” and “late” hematopoietic cytokines.Lowry et al., J. Cell Biochem. 58:410-415 (1995). “Early” stimulatoryfactors include SCF, FLT-3-L, IL-1, IL-3, IL-6, and IL-11. In additionto these positive regulators, hematopoiesis is also controlled byinhibitory cytokines. Negative regulation of myelopoiesis occurs throughseveral inhibitory cytokines, most notably MIP-1α (Cooper et al., Expt.Hematol. 22:186-193 (1994); Dunlop et al., Blood 79:2221-2225 (1992)),TGFβ3 (Jacobsen et al., Blood 78:2239-2247 (1991); Maze et al., J.Immunol. 149:1004-1009 (1992)) and TNFα (Mayani et al., Eur. J.Haematol. 49:225-233 (1992)).

Thus far a temporal change in these inhibitory peptides as a function oftime after cytotoxic therapy has not been quantitated. It is known,however, that under stressful conditions, such as irradiation,chemotherapy, blood loss, infection or inflammation, both stimulatoryand inhibitory growth factors play a major role in cellular adaptationprocesses. Cannistra et al., Semin. Hematol. 25:173-188 (1988). Understress, the quiescent CFU-S component of the stem cell compartment istriggered into active cell cycling and returns to the predominantly G₀G₁phase once normal bone marrow cellularity is restored. Becker et al.,Blood 26:296-304 (1965).

The recent literature has highlighted several important areas where anoninvasive method to monitor myelorecovery could have considerableclinical benefit. For example, to improve the safety and costeffectiveness of high-dose regimens, hematopoietic cell support(cytokines) has been used to accelerate marrow recovery followingmyeloablative therapy. This approach results in an earlier recovery ofperipheral blood counts, but the proliferative status of the marrowremains unknown and could be in a very active and sensitive state.

Another relevant example pertains to the use of allogeneic or autologousBMT, or more recently peripheral stem cell transplantation (SCT)following myelosuppressive or myeloablative therapy. Under thoseconditions, hematopoiesis is characterized by a prolonged and severedeficiency of marrow progenitors for several years, especially of theerythroid and megakaryocyte types, while the peripheral WBCs and PLTshave reached relatively normal values within a few weeks. Therefore,successful engraftment can not be measured by normalization of WBCs orPLTs, but requires another type of marker, perhaps one associated withnormal marrow stromal function. Domensch et al., Blood 85:3320-3327(1995). More information is needed to determine ‘true’ myelorecoverywhen either BMT or SCT is utilized. Talmadge et al., Bone MarrowTransplant. 19(2):161-172 (1997).

Yet, another area where a noninvasive measure of myelorecovery may beuseful is for scheduling leukapheresis. Since patient-to-patientvariability in time to marrow recovery is quite variable following G-CSFstem ell mobilization, it is difficult to predict the best time for thisprocedure. Identification of one or more markers of myelotoxic nadir andrecovery could advance SCT technology. Shpall et al, Cancer Treat. Res.77:143-157 (1997).

One investigator has shown that after allogeneic or autologous BMT, arise in endogenous G-CSF levels precedes and correlates with myeloidengraftment. Cairo et al., Blood 79(7): 1869-1873 (1992). Moreover, inpatients suffering from acute bacterial infections, whose rate ofmyelopoiesis must adapt to the enhanced demand, G-CSF, but not GM-CSF,was elevated. Selig et al., Blood 79:1869-1873 (1995). Additionalstudies demonstrated that the stem cell subset responsible forreconstitution is responsive to GM-CSF, IL-3, IL-6, and SCF. Wagemakeret al., Stem Cells 13:165-171 (1995). Other reports have quantified oneor more cytokines during a myelosuppressive episode. Sallerfors et al.,Br. J. Hematol. 78:343-351 (1991); Baiocchi et al., Cancer Research51:1297-1303 (1996); Chen et al., Jap. J. Clin. Oncol. 26:18-23 (1996).Heretofore, however, no one carefully studied the recovery phasefollowing myelosuppression, and there exists no correlation with theability to redose without severe toxicity. A relatively new stromalcell-produced positive stimulatory cytokine, FLT-3-L (Brasel et al.,Blood 88:2004-2012 (1996); Lisovsky et al., Blood 88(10):3987-97(1996)), has not been studied at all to date regarding eitherconstitutive or induced hematopoiesis. The ability to predict themagnitude of myelotoxicity in response to a given dose of RAIT wouldpermit patient-specific dosing. Red marrow absorbed doses have not beenhighly predictive of hematopoietic toxicity in RAIT-treated patients.DeNardo G L, DeNardo S J, Macey D J, Shen S, Kroger L A. Overview ofradiation myelotoxicity secondary to radioimmunotherapy using ¹³¹I-Lym-1as a model. Cancer. 1994; 73:1038-1048. Juweid M E, Zhang C, BlumenthalR D, Hajjar G, Sharkey R M, Goldenberg D M. Prediction of hematologictoxicity after radioimmunotherapy with ¹³¹I-labeled anticarcinoembryonicantigen monoclonal antibodies. J Nucl Med. 1999; 40:1609-1616.

Although the dose-toxicity relationship is likely to improve as morepatient-specific models for the calculation of red marrow dose areimplemented, more work needs to be done to define the tolerance ofpatients who have received therapy prior to nonmyeloablativeradioimmunotherapy (RAIT). Thus, methods need to be established thatreflect more accurately the marrow reserve in patients, so that theactivity prescription for RAIT can be adjusted accordingly.

In previous work (Blumenthal R D, Lew W, Juweid M, Alisauskas R, Ying Z,Goldenberg D M. Plasma FLT3-L levels predict bone marrow recovery frommyelosuppressive therapy. Cancer. 2000; 88:333-343), it was demonstratedthat 13% of the patient population studied experienced significantlyless toxicity than was predicted by their marrow dose and 15% of thesame population experienced significantly greater toxicity thanpredicted. Many of these patients have received multiple treatments ofexternal beam radiation therapy and/or chemotherapy prior to receivingRAIT. It was postulated that long-term hematopoietic damage from priorcytotoxic therapy might render a patient's marrow more “brittle” andtherefore more radiosensitive to the RAIT dose. Additionaltumor-produced cytokines may also be a significant factor influencingthe proliferation rate of marrow cells, thereby affecting their responseto radiation from RAIT. R. D. Blumenthal, A. Reising, E. Leon, and D. M.Goldenberg. Modulation of marrow proliferation and chemosensitivity bytumor-produced cytokines from syngeneic pancreatic tumor lines. AmericanSociety of Hematology Annual Meeting Abstracts, 2001; #946.

FLT3-L is a growth factor involved in early hematopoiesis, is expressedin transmembrane and soluble forms, and stimulates/co-stimulatesproliferation and colony formation of hematopoietic myeloid and lymphoidstem/progenitor cells (CFU-GM and CFU-GEMM) in bone marrow, spleen andperipheral blood. Lisovsky M, Braun S E, Ge Y, et al. Flt3-ligandproduction by human bone marrow stromal cells. Leukemia. 1996;10:1012-1018. Brasel K, McKenna H J, Morrissey P J, et al. Hematologicaleffects of flt3-Ligand in vivo in mice. Blood. 1996; 88:2004-2012.Papayannopoulou T, Nakamoto B, Andrews R G, et al. In vivo effects offlt3/flk2-ligand on mobilization of hematopoietic progenitors inprimates and potent synergistic enhancement with granulocytecolony-stimulating factor. Blood. 1997; 90:620-629. By itself, FLT3-Lhas weak colony-stimulating activity, but is additive togreater-than-additive on colony number and size when combined with othercolony stimulating factors (CSFs). In addition, a need still exists toestablish a predictive marker for a sizeable number of individuals whoexperience significantly less toxicity for a given marrow dose of RAIT,than was expected.

Therefore, a need exists in the art for improved methods, and kits forimplementing them, for predicting myelosuppressive recovery inconjunction with the foregoing deficient therapeutic techniques. Suchmethods could be used to help optimize treatment, informing theclinician of the appropriate timing of treatment, especiallyretreatment, thus avoiding toxic effects, while maximizing efficaciousones. Provided such a method, the art would posses new, optimizedmethods of treatment.

SUMMARY OF THE INVENTION

It is therefore an object of the invention to provide kits and methodsfor evaluating the myelosuppressive state of a patient. According tothis object, the invention provides a kit which contains at least onecytokine-specific detection reagent that is adapted to detect athreshold level a cytokine, which correlates with the myelosuppressivestate. In one embodiment, the cytokine specific reagent is specific forFLT3-L, TNF-α or TGF-β, and the reagent may comprise an antibody orantibody fragment.

Also according to this object of the invention, a method of assessingthe myelosuppressive state of a patient is provided. This method entailscomparing the amount of at least one cytokine in a patient sample with athreshold level, thereby gauging the myelosuppressive state of thepatient. In one embodiment, the cytokine specific reagent is specificfor FLT3-L, TNF-α or TGF-β, and the reagent may comprise an antibody orantibody fragment.

It is another object of the invention to provide an improved method oftreating cancer or any disease when using bone-marrow suppressiveagents. Further to this object, a method is provided where a patient isadministered an effective amount of an anti-cancer or other cytotoxicagent and the level of at least one cytokine is compared with athreshold level. In one embodiment, the cytokine is FLT3-L, TNF-α orTGF-β. In other aspects, the method involves using the threshold levelto guide treatment, so that when the threshold is approached or crossed,treatment is halted or decreased until it is no longer approached orexceeded

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Scattergram representation of 76 patients given different redmarrow doses of RAIT and the (a) percent platelet (PLT) loss; (b)percent WBC loss, (c) grade PLT toxicity and (d) grade WBC toxicityshown. All patients had normal initial pWBC and PLT counts at the timeof cytotoxic therapy (RAIT). {Open circles=normal toxicity; closedsquares=higher than expected toxicity and open triangles=lower thanexpected toxicity}. Twenty-three patients were omitted because theyeither had BM involvement on two or more known metastases or they hadhigher than normal values of either pWBCs (>10,000/mm³) or PLTs(>5.times. 10⁵/mm³).

FIG. 2. Plasma cytokine levels for SCF, FLT3-L, TNFα, TGFβ and MIP-1α(mean±SEM) for 5 untreated volunteers, for 14 patients derived from thenormal degree of toxicity group (open circles from FIG. 1), for 13patients with lower than expected toxicity (triangles in FIG. 1) and 12patients with higher than expected toxicity (solid squares in FIG. 1).

FIG. 3. Plasma FLT3-L levels (mean±SEM) for patients sorted by gradetoxicity—those <grade 3 (27-28 patients) and those >grade 3 toxicity(11-12 patients). Average RM dose for all groups is noted in the base ofthe vertical bars and significance (t-test) noted at the top of the bar.

FIG. 4. Comparisons of platelet nadir (PN) with FLT3-L adjustedpredictors of toxicity.

FIG. 5. Comparisons of 1/platelet nadir (PN) with FLT3-L adjustedpredictors of toxicity.

DETAILED DESCRIPTION OF THE INVENTION

A. Definitions

As used herein, “myelosuppression” refers to the suppression of one ormore components of hematopoiesis, which manifests in aberrant levels ofone or more of the cell types that are the products of this process. Fora review of hematopoiesis, and characteristics of hematopoietic cells,see CLINICAL IMMUNOLOGY: PRINCIPLES AND Practice, Vol. 1, Ch. 2, pp.15-24 (Lewis and Harriman, eds. Mosby—Year Book, Inc. 1996), which pagesare hereby incorporated by reference. On a general level it refers todecreases in white blood cell (WBC) and/or platelet counts. It alsorefers, on a more specific level, to suppression of one or more of thefollowing cells that result from hematopoiesis: B-cells, T-cells,natural killer cells, dendritic cells, macrophages, neutrophils,eosinophils, basophils, mast cells and platelets. On the other hand,therefore, “myelorecovery” is the opposite of myelosuppression.

As used herein, a “stimulatory cytokine” is one that promoteshematopoiesis at one or more stages of differentiation. Stimulatorycytokines include SCF, FLT-3-L, IL-1, IL-3, IL-6, IL-11, and othersknown to the skilled artisan.

As used herein an “inhibitory cytokine” has a negative effect on one ormore stages of hematopoiesis. Exemplary inhibitory cytokines includeMIP-1α, TGFβ3, TNFα and others known in the art.

As used in a general sense herein, unless otherwise indicated bycontext, the term “antibody” includes “antibody fragment” and otherforms of reengineered antibody subfragments which retain the ability tobind to the specific antigen to which they were developed.

B. Principles of the Invention

The invention relates to the ability to predict myelorecovery after asubject experiences myelosuppression (e.g., after radiation, cytotoxicchemotherapy, or other means) by monitoring various inhibitory andstimulatory cytokines. The present inventors have discovered thatthreshold levels of certain cytokines can be used to guide thehealth-care professional in using myelosuppressive therapies. Inparticular, these threshold levels provide a marker, indicating whetheror not a patient will tolerate such therapy. A common application is inmonitoring cytoreductive therapies, where the subject threshold levelsare used to decide whether a patient is sufficiently recovered from onedose of a myelosuppressive agent to tolerate another, perhaps increaseddose.

The cytokine levels monitored in the inventive methods include theso-called “early” stimulatory cytokines and the inhibitory cytokines. Tobe useful in these methods and kits, a statistically significantthreshold level of the cytokine (or a combination of them) thatcorrelates with myelosuppressive recovery is ascertainable. The artisanwill be familiar with such statistical analysis and may readilyascertain such threshold levels, as demonstrated below in the Examples.

In a broad sense, a threshold level may be a level that is found in anormal volunteer, any deviation associated with myelosuppression beingindicative of that state. In particular, the threshold level should beset such that specificity [(true negative) divided by (true negativeplus total population)], accuracy [(true positive plus true negative)divided by (total population)] and sensitivity [(true positive) dividedby (true positive plus false negative)] are maximized. The artisan willrecognize, however that such maximization often represents a trade-off,since higher specificity, accuracy or sensitivity can result in theothers being lowered. Some inventive methods yield greater than about65% specificity, accuracy and sensitivity, while some preferred methodsyield at least about 75% specificity, accuracy and sensitivity.

“Early” stimulatory factors include, but are not limited to, SCF,FLT-3-L, IL-1, IL-3, IL-6, and IL-11. These factors are thought to beinvolved in the early stages of myelorecovery. Thus, when they arepresent, the damage should be at its worst. Accordingly, a statisticallysignificant threshold should be ascertainable, which, when exceeded,counsels against continued therapy or indicates reducing the dose.

Inhibitory cytokines, in contrast, likely are present whenmyelosuppressive recovery is virtually complete, when the process isturning itself off. Hence, the threshold level for these cytokines willrepresent a minimum level, below which therapy should be reduced orhalted. Exemplary inhibitory cytokines include MIP-1α, TGFβ3 and TNFα.

The invention also contemplates the usefulness of trends in predictingmyelorecovery. Thus, it is possible that the absolute amount of plasmacytokine needs to be coupled with the duration since the cytokinereached its peak. For example, as seen below in the working examples,since the values for FLT3-L ranged from below 100 pg/ml to over 400pg/ml, it is possible that readings of FLT3-L in or just above thenormal range may need to be evaluated again a few days later todetermine whether plasma FLT3-L is on the rise, or is returning backdown to baseline levels after being elevated. It is contemplated thatthose patients whose FLT3-L levels have returned to normal andmaintained a normal baseline level for several weeks can tolerate higherdoses than patients who have recovered only days earlier from amyelosuppressive episode and elevated FLT3-L. This provides anexplanation of the low toxicity group that does not strictly correlatewith any of the cytokines measured.

C. Kits of the Invention

The kits according to the invention typically comprise at least onecytokine-specific detection reagent. Some kits contain at least twocytokine-specific detection reagents. In most cases, each reagent willbe adapted to detect a threshold level of cytokine, which correlateswith the myelosuppressive state of a patient. In one aspect, theinvention contemplates a kit for assessing the myelosuppressive state ofa patient, which is useful in guiding the physician in choosing anoptimal treatment regimen. They may be applied, for example, to monitormyelosuppressive treatments, to monitor efficacy of myelostimulatorytreatments and to monitor recovery from myelosuppressive disorders.

Some embodiments of the present kits contain the detection reagent inassociation with a suitable testing substrate. Suitable substratesinclude “dipsticks,” test-strips, microtiter plates, microscope slides,and the like. The kits of the invention generally implement the methods,described below, and should be read in that context.

Cytokine-Specific Detection Reagents

The cytokine-specific detection reagent of the kit generally confers theability to detect specifically the cytokine of interest, in some casesquantitatively. Typically this reagent will be able to bind specificallyto a cytokine, and will be detectable, directly or indirectly. Forinstance, the reagent may be an antibody, and may comprise a detectablelabel, such as a radionuclide, an enzyme or a fluorescent tag. The labelmay be detected, for example, using conventional immunoassays, whichinclude enzyme-linked immunosorbant assays (ELISAs), radioimmunoassays(RIAs), and the like. Suitable methods can be found in CURRENT PROTOCOLSIN MOLECULAR BIOLOGY, Chapter 11 (Ausubel et al., eds., John Wiley &Sons, Inc. 1997), which is hereby incorporated by reference.

Due to their recognized ability to bind specifically and to their easeof production, antibodies are contemplated as a means of conferring thecytokine-binding ability of the detection reagent. Antibodies include,but are not limited to polyclonal antibodies, monoclonal antibodies(mAbs), humanized or chimeric antibodies, single chain antibodiesincluding single chain Fv (scFv) fragments, Fab fragments, F(ab′)₂fragments, fragments produced by a Fab expression library,epitope-binding fragments, and multivalent forms of any of the above.

In general, techniques for preparing polyclonal and monoclonalantibodies as well as hybridomas capable of producing the desiredantibody are well known in the art (Campbell, MONOCLONAL ANTIBODYTECHNOLOGY: LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY,(Elsevier Science Publishers 1984); St. Groth et al., J. Immunol.Methods 35:1-21 (1980); Kohler and Milstein, Nature 256:495-497 (1975)),the trioma technique, the human B-cell hybridoma technique (Kozbor etal., Immunology Today 4:72 (1983); Cole et al., in MONOCLONAL ANTIBODIESAND CANCER THERAPY, Alan R. Liss, Inc. (1985), pp. 77-96). Affinity ofthe antisera for the antigen may be determined by preparing competitivebinding curves, as described, for example, by Fisher, Chap. 42 in:MANUAL OF CLINICAL IMMUNOLOGY, 2d ed., (Rose and Friedman, eds., Amer.Soc. For Microbiology 1980).

Antibody fragments include any portion of the antibody which includesthe paratope and is capable of binding a cytokine of interest. Antibodyfragments specifically include F(ab′)₂, Fab, Fab′ and Fv fragments.These can be generated from any class of antibody, but typically aremade from IgG or IgM. They may be made by conventional recombinant DNAtechniques or, using the classical method, by proteolytic digestion withpapain or pepsin. See CURRENT PROTOCOLS IN IMMUNOLOGY, chapter 2,(Coligan et al., eds., John Wiley & Sons 1991-92).

F(ab′)₂ fragments are typically about 110 kDa (IgG) or about 150 kDa(IgM) and contain two antigen-binding regions, joined at the hinge bydisulfide bond(s). Virtually all, if not all, of the Fc is absent inthese fragments. Fab′ fragments are typically about 55 kDa (IgG) orabout 75 kDa (IgM) and can be formed, for example, by reducing thedisulfide bond(s) of an F(ab′)₂ fragment. The resulting free sulfhydrylgroup(s) may be used to conveniently conjugate Fab′ fragments to othermolecules, such as localization signals.

Fab fragments are monovalent and usually are about 50 kDa (from anysource). Fab fragments include the light (L) and heavy (H) chain,variable (V_(L) and V_(H), respectively) and constant (C_(L) C_(H),respectively) regions of the antigen-binding portion of the antibody.The H and L portions are linked by one or more intramolecular disulfidebridges.

Fv fragments are typically about 25 kDa (regardless of source) andcontain the variable regions of both the light and heavy chains (V_(L)and V_(H), respectively). Usually, the V_(L) and V_(H) chains are heldtogether only by non-covalent interactions and, thus, they readilydissociate. They do, however, have the advantage of small size and theyretain the same binding properties of the larger Fab fragments.Accordingly, methods have been developed to crosslink the V_(L) andV_(H) chains, using, for example, glutaraldehyde (or other chemicalcrosslinkers), intermolecular disulfide bonds (by incorporation ofcysteines) and peptide linkers. The resulting Fv is now a single chain(i.e., scFv).

Antibodies also include single chain antibodies and fragments (U.S. Pat.No. 4,946,778; Bird, Science 242:423426 (1988); Huston et al., Proc.Natl. Acad. Sci. USA 85:5879-5883 (1988); and Ward et al., Nature334:544-546 (1989)). Single chain antibodies are formed by linking theheavy and light chain fragments of the Fv region via an amino acidbridge, resulting in a single chain FV (scFv).

Some exemplary kits contain at least one cytokine-specific reagent thatis specific for FLT3-L, TNF-α or TGF-β. In one aspect of the invention,the reagent comprise an enzyme-linked antibody or antibody fragment.

Adapting the Reagent to Detect a Threshold

The kits of the invention detect a specific threshold of cytokine, whichcorrelates with the myelosuppressive state of the patient. Suchthresholds, and their determination, are detailed below. Forconvenience, it is advantageous to adapt the cytokine-specific detectionreagent(s) to detect a certain threshold. In this way, a “yes” or “no”answer can be provided, generally indicating whether the patient ismyelosuppressed or not. Thus, for example, colorimetric detection mightbe employed, whereby the presence of color indicates that a thresholdlevel, correlating with the myelosuppressive state, has been reached.

Typically, the reagents of various assays (e.g., ELISAs, RIAs, RT-PCRs,and the like) will be able to detect levels of the target cytokine(s)that are lower than the threshold, i.e., they are more sensitive thanthey need to be. The artisan will be well-aware of methods of reducingthe sensitivity of the present systems in order to provide a signal at agiven threshold level. A particularly useful kit will include a reagentsystem that can provide a “yes” or “no” answer as to whether a patienthas recovered sufficiently from myelosuppression to tolerate furthercytotoxic therapy.

Using the Kits of the Invention

The kits may be adapted for private to commercial-scale use, for theconvenience of the individual clinician, the clinical research centerand even commercial diagnostic laboratories. For example, in a privateclinical setting, a “dipstick”-type arrangement may be convenient. Inone aspect, the cytokine-specific detection reagent may come applied tothe dipstick. Thus, the kit may be used by contacting a patient sampleto the dipstick-associated reagent. The detection reagent may then bevisualized using conventional colorimetric means, for example. Ofcourse, another arrangement may call for contacting the sample with thedipstick, and then application of the cytokine-specific detectionreagent; the exact arrangement is a matter of choice.

In another example especially suitable for larger laboratories, the kitscan be implemented in microtiter plates (e.g., 96-well plates). The samearrangement of reagents would apply, where the detection reagent iseither supplied in the plate or is added after the sample is applied tothe plate. In any event, given the availability of high-throughputreaders for microtiter plates, very large numbers of samples could behandled automatically in this manner. Again, specific arrangements are amatter of design choice.

Methods of the Invention

The invention provides a general method of assessing themyelosuppressive state of a patient. The basic method comprisescomparing the amount of at least one cytokine to a threshold level. Themyelosuppressive state of the patient is then gauged relative to thatthreshold. The cytokines monitored, as explained above, may be earlystimulatory or inhibitory cytokines, or combinations thereof. In oneaspect, the method involves at least monitoring levels of FTL3-L.

When plasma samples are used in the present methods, it is advisable toassure the amounts measured are a function of marrow cell production,and not peripheral blood cell or tumor cell production. Fortunately,peripheral blood cells by themselves are unable to produce mostcytokines. In fact, PCR amplification of reverse-transcribed RNA fromperipheral blood cells in healthy individuals reveals that TGFβ, MIP-1αand IL-1β were expressed, but that SCF, IL-6, G-CSF, GM-CSF, IL-1α werenot expressed. Cluitmans et al., Ann. Hematol. 75(1-2):27-31 (1997).Moreover, tumor-produced cytokines may confound marrow-producedcytokines. Several cytokines including TGFβ and TNFα are elevated inblood samples from ovarian, cervical, and endometrial cancer patients.Chopra et al., J. Cancer Res. Clin. Oncol. 123:167-172 (1997); Chopra etal., Cancer J. Sci. Am. 2:279-285 (1996); Chopra et al., CancerInvestigation 16(3):152-159 (1998). However, there is no indicationwhether this is true for all cancer types or that there is any evidencethat FLT3-L, SCF, or MIP-1α are produced by tumors. The artisan willreadily understand how to test and control for marrow-derivedproduction.

The inventive methods may be used in conjunction with conventionaltherapies that induce myelosuppression, or where subjects have beenexposed to ionizing radiation. Thus, where the threshold level isapproached or crossed, therapy generally will be halted or reduced. If apatient is then re-tested, and this test indicates that the threshold isno longer approached or crossed, therapy may resume. On the other hand,where a patient is being treated, and the inventive test indicates thatthe threshold has not been approached or crossed, the next therapeuticdose may be administered safely. In this manner, dosing regimens may beinformed by constant monitoring, increasing dose and frequency untilthreshold levels are approached or crossed, at which point dosing may bedecreased or eliminated. In this context, a threshold level isapproached when a cytokine level is within at least about 15% of thethreshold number, but preferably is within at least about 10% of thethreshold.

Preferred cytokines for monitoring in the present methods includeFLT3-L, TNF-α and TGF-β. Since FLT3-L is an early stimulatory cytokine,the relevant threshold is a maximum. On the other hand, since TNF-α andTGF-β are inhibitory cytokines, the relevant threshold is a minimum.Exemplary threshold levels include: at least about 135 pg/ml of plasmafor FTL3-L; at most about 0.5 pg/ml of plasma for TNF-α; and at mostabout 15 pg/ml of plasma for TGF-β. Again, it is not only these absolutethresholds that are important; the artisan will also recognize thattrends toward these thresholds are significant in prediction, especiallywhen viewed over a multi-day (1-3) temporal window.

One aspect of the invention contemplates a method of treating cancerthat involves administering to a patient in need of treatment, aneffective amount of an anti-cancer agent and using the presentmyelorecovery monitoring techniques to inform treatment, especiallydosing. Thus, cytokine levels may be evaluated at intervals throughouttreatment, beginning before or after the first administration of ananti-cancer agent.

Conventional anti-cancer agents include chemotherapeutics andradiation-based therapies. Chemotherapeutic agents include alkylatingagents, antimetabolites, various natural products (e.g., vincaalkaloids, epipodophyllotoxins, antibiotics, and amino acid-depletingenzymes), and taxanes. Specific classes of agents include nitrogenmustards, alkyl sulfonates, nitrosoureas, triazenes, folic acidanalogues, pyrimidine analogues, purine analogs, platinum complexes,adrenocortical suppressants. Some exemplary compounds includeactinomycin, cyclophosphamide, chlorambucil, CPT-11, methotrexate,fluorouracil, cytarabine, thioguanine, vinblastine, vincristine,doxorubicin, daunorubicin, mitomycin, cisplatin, hydroxyurea, taxols,and platinum compounds, including oxaliplatin. Suitable chemotherapeuticagents are described in REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed.(Mack Publishing Co. 1995), and in GOODMAN AND GILMAN'S THEPHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed. (MacMillan Publishing Co.1985), as well as revised editions of these publications, incorporatedherein in their entirety by reference. Other suitable chemotherapeuticagents, such as experimental drugs, are known to those of skill in theart. The known dosing protocols for these drugs may be optimized usingthe present methods of evaluating myelosuppression.

The invention provides a method of assessing the state of the bonemarrow of a patient, comprising comparing the amount of at least onehematopoietic cytokine in a sample from the patient with a thresholdlevel, thereby gauging the state of the bone marrow of a patient. In oneembodiment of the invention, the patient is in a myelosuppressive state.Preferably, the hematopoietic cytokines are FLT3-L, TNF-α and TGF-β.

In another embodiment, the present invention provides a method ofassessing myelorecovery in a patient comprising repeatedly assessing thestate of the bone marrow in a patient while the patient is undergoingsuccessive treatments of myelosupressive therapy.

The invention further provides a method of predicting the bone marrowtoxicity dose delivered to a subject. Preferably, the FLT3-L levels in asubject's blood or plasma are determined and the calculated bone marrowradiation dose is adjusted according to the plasma or blood level ofFLT3-L in the subject. In one embodiment of the present invention, priorto measuring plasma or blood level of FLT3-L in the subject, the subjectwas given cytotoxic chemotherapy and/or radioimmunotherapy. In anembodiment of the present invention, such cytotoxic chemotherapy isionizing radiation which was delivered by radioimmunotherapy.

The FLT3-L levels in a subject can be measured at least once butadditional measurements of FLT3-L levels are also contemplated topredict whether the FLT3-L levels are on the rise or in the process offalling from their peak. Preferably, the FLT3-L plasma or blood levelsare measured at least once before and once after potentialmyelosuppressive therapy.

Bone marrow radiation dose can be determined by a variety of methods.Specifically, a pretherapy tracer study performed before treatment ofthe subject can be used to determine the red marrow radiation dose.Preferably, the pretherapy tracer study can be performed 1-2 weeks priorto treatment. Specifically, after a subject is given a diagnosticantibody activity infusion, blood cumulated activity concentrations andtotal body cumulated activities can be determined. Preferably, bloodcumulated activity concentrations are determined by counting samples ofwhole blood in a calibrated gamma well counter to obtain blood activityconcentrations at various time points after the end of the antibodyactivity infusion. These time-activity concentration curves can beanalyzed to determine the slopes of the distribution and eliminationphases and their respective intercepts. Preferably, a nonlinear leastsquares curve-fitting algorithm is used to fit the curve. These curvescan then integrated to obtain the blood cumulated activityconcentration. Total body cumulated activities can also be determined.Preferably, total body cumulated activities can be determined usingeither whole-body gamma camera counts or handheld radiation probe countsobtained at multiple time points post-administration. Additional methodsof determining blood cumulated activity concentrations and total bodycumulated activities are readily apparent to one of skill in the art andare encompassed by the present invention.

Additional methods can be used to characterize the red marrowbiokinetics, including determination of the red marrow cumulatedactivity from scintillation camera image-based analyses (Siegel J A, LeeR A, Pawlyk D A, Horowitz J A, Sharkey R M, Goldenberg D M. Sacralscintigraphy for bone marrow dosimetry in radioimmunotherapy. Nucl MedBiol. 1989; 16:553-559), compartmental modeling techniques (Loh A,Sgouros G, O'Donoghue J A, et al. Pharmacokinetic model ofiodine-131-G250 antibody in renal cell carcinoma patients. J Nucl Med.1998; 39:484-489), or use of magnetic resonance spectroscopy to providea patient-specific estimate of the red marrow extracellular fluidfraction (Ballon D, Jakubowski A, Gabrilove J, et al. In vivomeasurements of bone marrow cellularity using volume-localized protonNMR spectroscopy. Magnetic Reson Med. 1991; 19:85-95).

The invention further provides a method of determining the dose ofmyelosuppressive treatment delivered to the bone marrow in a subject bymeasuring the level of FLT3-L in the subject; using the ratio of FLT3-Lin the subject to the level in normal subjects to adjust the dose ofmyelosuppressive treatment delivered to the bone marrow. In oneembodiment of the invention, the myelosuppressive treatment is cytotoxicchemotherapy or radioimmunotherapy.

In an embodiment of the present invention, the FLT3-L level in a normalsubject is from about 40 pg/mL to about 85 pg/mL, and most preferablythe FLT3-L levels are about 80 pg/mL.

Bone marrow radiation dose determined by the above methods is then usedto determine a treatment activity prescription.

EXAMPLES Example 1 Methods of Use for Practicing the Invention

Patient Population and Collection of Patient Blood.

Solid tumor patients enrolled in Institutional Review Board-approvedGarden State Cancer Center clinical radioimmunotherapy (“RAIT”) trialshave had multiple cycles of previous chemotherapy using various drugs(e.g., doxorubicin, methotrexate, topotecan,cyclohexyl-chloroethylnitrosourea (CCNU), mitomycin, etc.) and differentdurations ranging from 1 to 24 months since their previous treatment.Juweid et al., Cancer 80:2749-2753 (1997). Patient blood (3 ml) wascollected on the day of scheduled radioimmunotherapy into citrate-tubesand complete blood counts (CBCs) were performed to establish pWBC andPLT counts. Blood was collected every 3-7 days after RAIT and themaximum percent loss, and toxicity grade for both WBCs and PLTs weredetermined.

Plasma Cytokine Immunoassays.

Plasma FLT3-L, SCF, and TGF-β in patient blood samples were measured byR&D Quantikine Immunoassay kits (Minneapolis, Minn.). These assaysemploy a quantitative sandwich enzyme immunoassay. The optical density(OD) at 570 nm is subtracted from the OD at 450 nm to correct for plateimperfections. Average duplicate readings for each sample are read froma linear standard curve. TNFα and MIP-1α were analyzed by CYTImmuneSciences' competitive enzyme immunoassay kits (College Park, Md.),resulting in an inverse relationship between OD and concentration. Thekits use an amplified color generation system in which the alkalinephosphatase reaction provides a cofactor that initiates a redo cyclingreaction leading to the formation of a colored (formazan) red product.The OD was read at 492 nm. All assay kits have high sensitivity, arespecific, and show no significant cross-reactivity with any other murineor human cytokine.

Red Marrow Dosimetry.

The red marrow dose was estimated in all patients from the cumulatedactivity in the blood based on the blood clearance data, and taking intoaccount the contribution from the whole body activity. The use of amarrow/blood activity concentration ratio of 0.36 was used, which isconsistent with the recommendations of the Dosimetry Task Group of theAmerican Association of Physicists in Medicine. Siegel et al., AntibodyImmunoconj. Radiopharm. 3:213-233 (1990); Fisher et al., Cancer73:905-911 (1994); Sgouros et al., J. Nucl. Med. 34:689-694 (1993). Thecorrected blood activity concentration was always multiplied by 1,500,the weight in grams of the marrow in an average adult. The mean dose incGy was then obtained according to the MIRD schema, taking into accountthe contribution from the whole body activity. Loevinger et al., Soc.Nucl. Med. (1976); Cloutier et al., J. Nucl. Med. 14:53-55 (1973).

Toxicity Assessment.

Myelotoxicity was graded according to the National Cancer Institute(NCI) toxicity criteria. All patients given therapeutic doses werefollowed for hematological toxicity by monitoring CBCs weekly. In case agrade 2 thrombocytopenia or leukopenia developed, biweekly measurementswere taken, and in the case of grade 3 or 4 thrombocytopenia orleukopenia, measurements were taken every other day until the nadir hadbeen determined. The patient's blood counts were followed until completehematological recovery was established.

Statistical Analysis.

Single factor analysis of variance (F-test) was performed on serumcytokine measurements in normal volunteers, chemotherapy naive cancerpatients, and cancer patients with either normal levels,lower-than-expected levels, or higher-than-expected levels ofmyelosuppression for their given RM dose. The ability of a single markeror a combination of serum cytokine markers to predict myelosuppressiveresponses was determined using the following formula:Sensitivity=[TP/(TP+FN)]; specificity=[TN/(TN+FP)]; andaccuracy=[(TP+TN)/(TP+TN+FN+FP)], where TP=true positive; TN=truenegative; FP=false positive; and FN=false negative.

In a true-positive, a stimulatory-cytokine is elevated and/or aninhibitory cytokine is below normal and the patient experienceshigh-toxicity. A true-negative means the stimulatory cytokines and/orthe inhibitory cytokines are normal and toxicity is within normallimits. A false-positive means a stimulatory cytokine is elevated and/oran inhibitory cytokine level is below normal, but the magnitude oftoxicity is within the expected range or low. A false-negative meansstimulatory and/or inhibitory cytokines are within normal limits, buttoxicity is high and could not be predicted. An alternative clinicallyuseful measure to express test efficiency is the likelihood ratio tocharacterize behavior of the diagnostic test. The positive likelihoodratio (LR+) is defined as the ratio of sensitivity over (1-specificity).When it exceeds 1, the odds favoring positive diagnosis increase, and asit approaches 1, the test is indeterminate. The negative likelihoodratio (LR−) is defined as (1-sensitivity) over specificity. Simel etal., J. Clin. Epidemiol. 44:763-770 (1991).

Example 2 Ascertaining a Statistically Significant Threshold Level of aGiven Cytokine

Seventy-four solid-tumor patients were selected from an initialninety-nine patients by omitting all individuals with bone marrowmetastases and all patients with an initial WBC or PLT count that wasunusually high (>10,000 WBC/mm³ or >550,000 PLT/mm³). All patients wererefractory to chemotherapy and entered clinical RAIT trials at ourresearch center. The RM dose delivered from the therapeutic dose wascalculated for each person. WBC and PLT toxicity were determined at thenadir as the percent loss from the initial count (upper panels) or asgrade toxicity (lower panels), and the results plotted against the RMdose (FIG. 1). The majority of patients (52-56 out of 74 for percentloss and 40-44 out of 74 for grade toxicity) conformed to a well-definedlinear relationship between RM dose and toxicity (O).

However, some patients (8 to 13) clearly exhibited less toxicity thanwas expected, given their RM dose (Δ) and other patients experiencedmuch greater toxicity (9 to 15) than most other patients did (●). Usingpercent loss, only 5 individuals who did not fit the linear patterndeviated for both WBCs and PLTs, 2 with excess toxicity for both and 3with less-than-expected toxicity for both. Thirteen had excess PLTtoxicity with normal WBC toxicity and 5 had excess WBC toxicity andnormal PLT toxicity. Using grade of toxicity as a criterion, 7individuals deviated from expectations; 2 with excess WBC and PLTtoxicity and 5 with less toxicity for both than was expected. Anadditional 8 patients had excess PLT toxicity with normal WBC toxicityand 7 had excess WBC toxicity, but normal PLT toxicity (Table 1A).

Since an excess toxicity of either WBC or PLT becomes dose-limiting, allpatients who deviated even in one category would benefit fromavailability of a marker to predict excess-toxicity. Of thoseindividuals with excess PLT toxicity (15 with excess % loss and 10 withexcess grade), 9 were elevated for both, only 1 has excess grade but anormal % loss and 5 had an excess % loss, but a normal grade toxicity.Of those individuals with excess WBC toxicity (9 with excess % loss and11 with excess grade toxicity), 5 were high for both parametersmeasured, and 6 were high for grade toxicity but had a normal % loss,and 3 had a high % loss but a normal grade toxicity (Table 1B). Ifpatients demonstrate a high initial WBC and/or PLT count on the day ofRAIT (upper end of normal range), they could conceivably experience ahigh percent loss but a reasonable grade toxicity. If WBC and/or PLTcounts start out at the low end of the normal range on the day of RAIT,then the patient may experience a high grade toxicity but not a highpercent loss. TABLE 1 Summary of the Number of Patients with AbnormalDegree of Toxicity in Response to RAIT (based on scattergram in FIG. 1)A. Effect on WBCs and/or PLTs Both WBCs and WBCs only PLTs only PLTsEffected Effected Effected Measurement: High Low High Low High LowPercent Loss 2 3 5 6 13 5 Grade Toxicity 2 5 9 6 8 8 Note: Total of 74patients included in the analysis. B. Percent Loss and Grade ToxicityExcess Grade- Excess % Excess % Excess Excess- Normal % Loss -- Normal NLoss Grade Both Loss Grade Excess PLT 16 15 10 9 1 6 Toxicity Excess WBC15 11 9 5 6 4 Toxicity Note: All patients has multiple cycles ofchemotherapy between 1 and 24 mo. prior to entering these RAIT clinicaltrials.

From the patients described, thirty-nine individuals were selected andsorted them into three subgroups, the first showing “normal” WBC and PLTtoxicity (N=14), the second showing low toxicity (N=13), and the thirddemonstrating “high” WBC or PLT toxicity (N=12). As shown in Table 2,the three groups received similar RM doses (139±28 vs. 190'32 vs. 141±51cGy, respectively). All three groups had similar initial WBC(6,000±2,000/mm³ in the first group vs. 8,000±2,000/mm³ in the lattertwo groups) and initial PLT counts (280,000±112,000/mm³ vs.233,000±84,000/mm³ vs. 203,000±65,000/mm³, respectively). The groupreferred to as excess toxicity had a significantly higher PLT loss(81±11% vs. 54±20% in the normal toxicity group; p<0.001) and Grade PLTtoxicity (3±1 vs. 1±1; p<0.001). The group also had a higher grade WBCtoxicity (2±1 vs. 1±1; p<0.05). TABLE 2 Patient Group Characteristicsfor Cytokine Marker Studies. Normal Toxicity Low Toxicity ExcessToxicity Variable (N = 14) (N = 13) (N = 12) Months Post Chemotherapy 4± 6 7 ± 9 5 ± 4 (range: 2 to 18) (range: 2 to 24) (range: 1 to 13) RMDose (cGy) 139 ± 28  190 ± 32  141 ± 51  Initial pWBC Count/μl (×1000) 6± 2 8 ± 2 8 ± 2 Initial PLT Count/μl (×1000) 260 ± 112 233 ± 84  203 ±65  % pWBC Loss Post RAIT 45 ± 21 42 ± 18 52 ± 25 (p < 0.1 = NS) * % PLTLoss Post RAIT 54 ± 20 43 ± 14 81 ± 11 (p < 0.06) (p < 0.001) Grade pWBCToxicity Post RAIT 1 ± 1 0 ± 1 2 ± 1 (p < 0.06) (p < 0.05)  Grade PLTToxicity Post RAIT 1 ± 1 0 ± 0 3 ± 1 (p < 0.01) (p < 0.001)* p values are relative to normal toxicity group

We tested five cytokines in patient plasma (FIG. 2) for statisticalcorrelation to myelorecovery. Table 3 shows the cytokines tested and thecharacteristics of the assays used. TABLE 3 Characteristics of CytokineImmunoassays FLT3-L SCF TGFβ1 TNFα MIP-1α Sensitivity 7 pg/ml 9 pg/ml 7pg/ml 0.2 ng/ml 0.2 ng/ml Linearity (range %) 105% 104% 013% — — Range0-1000 pg/ml 0 to 2000 pg/ml 0 to 2000 pg/ml 0.2 to 50 ng/ml .2 to 50ng/ml Recovery 94-110% 84-112% 94-110% — 85-104% Normal Values 58.6 to130.9 1000 to 1790 15.6 to 32.4 1.0 to 5.0 15 to 46 Cross-reactivity — —TGF-β5-1.5% — — TGF-β3-0.9%

SCF was similar in all groups studied; 1498±136 pg/ml vs. 1311±119 pg/mlvs. 1177±102 pg/ml for the normal, low, and high toxicity groupscompared with 1138±183 pg/ml in untreated volunteers and 1060±217 pg/mlfor cancer patients who received no prior chemotherapy. In contrast toresults with SCF, the other stimulatory cytokine, FLT3-L, showed asignificant elevation in the excess-toxicity group (235±29 pg/ml;p<0.001) compared with patients with normal or low toxicity (81±11 pg/mland 79±12 pg/ml, respectively), or with untreated volunteers (52±6pg/ml).

Surprisingly, while a reduction in inhibitory cytokines was postulatedto exist in the excess toxicity group, instead plasma TNFα was higher inthe excess toxicity group. This was not significantly different,however, from other groups because of a marked variability betweenpatients (2.62±1.03 compared with normal toxicity patients 1.50±0.33pg/ml or 1.80±0.54 pg/ml in the untreated volunteers). Plasma TGFβ wasalso not significantly different (28.1±4.9 pg/ml in the excess toxicitygroup and 38.1±7.5 in the normal toxicity group). Plasma MIP1α wassignificantly lower in the excess toxicity group (2.23±1.09 pg/ml vs.5.08±0.91 pg/ml in the normal toxicity group and 5.10±1.80 in theuntreated volunteers group; p<0.05). However, the low toxicity groupalso had reduced plasma MIP1α (2.47±0.68 pg/ml; p<0.05).

Of the five plasma cytokines evaluated, FLT3-L was found to be mostinformative regarding anticipated toxicity as a function of RM dose.Moreover, If patient data are sorted according to toxicity grade (<grade3, or ≧grade 3) independent of RM dose, instead of sorting patient databy normal-, low-, or high-toxicity, the importance of FLT3-L becomeseven stronger (FIG. 3). Of thirty-nine patients, 27 had <grade 3 PLTtoxicity and 12 had ≧grade 3 PLT toxicity (the numbers are 28 and 11patients for WBC toxicity, respectively). The RM doses for these groupswere similar. Plasma FLT3-L (mean±SEM) for PLT toxicity <grade 3 was84.4±8.8 pg/ml and 220.6±35.7 pg/ml for PLT toxicity ≧grade 3 (p<0.001).A similar tendency existed for WBC toxicity, but was not statisticallysignificant.

The usefulness of FLT3-L alone or in combination with other plasmacytokine measurements to predict high-toxicity is presented in Table 4.Stimulatory cytokine levels were set above the upper normal limit andinhibitory cytokine levels were set below the lower normal limit, bothspecified in Table 3. Results are expressed as sensitivity, specificity,and accuracy; the latter measurement permits identification of both thetrue positives and true negatives from the total population. Of all 7permutations evaluated, high FLT3-L levels alone (>135 pg/ml) resultedin the best values for sensitivity =0.83 (one-sided 95% confidenceinterval is 0.66-1). Likewise, the accuracy is 0.85 and the specificityis estimated at 0.89 (95% confidence interval being 0.79-1). Combiningelevated FLT3-L levels with low TNFα or low MIP1α resulted in maximumspecificity, but dramatically reduced both sensitivity (0.10 or 0.56,respectively) and accuracy (0.29 and 0.25, respectively). Alternatively,adjusting the threshold for FLT-3 to 170 pg/ml results in a reducedsensitivity of 0.62, but an increased specificity of 1.0 and nosignificant change in accuracy (0.87) compared with a FLT3-L cutoff of135 pg/ml. Thus, the threshold set for FLT3-L will determine whethersensitivity or specificity is higher. By using the lower FLT3-Lthreshold of 135 pg/ml, the positive and negative likelihood ratios canbe calculated as a means of expressing predictability of FLT3-L as adiagnostic test. The estimated positive likelihood ratio is 7.5 with a95% confidence interval 2.5-22.5. The negative likelihood ratio is 0.19,with a 95% confidence interval of 0.05-0.67. TABLE 4 Ability of SerumCytokines to Predict Thrombocytopenia * Sensitivity Specificity AccuracyHigh FLT3-L (>135 pg/ml) 83% 89% 85% High FLT3-L or Low 69% 84% 78% TNFα(<0.5 pg/ml) High FLT3-L or Low 69% 85% 77% TGFβ (<15 pg/ml) High FLT3-Lor Low 50% 83% 75% TNFα or Low TGFβ High FLT3-L and Low 10% 100%  29%TNFα High FLT3-L or Low 63% 81% 34% MIP-1α (<10 pg/ml) High FLT3-L andLow 56% 100%  25% MIP1α* N = 39; 12 with high toxicity; 13 with low toxicity; 14 with normaltoxicity for the given RM dose.

Hematopoiesis proceeds under the influence of early and late stimulatoryand inhibitory cytokines (Cannistra et al., Semin. Hematol. 25:173-188(1988); Whetton et al., Biochem. Biophys. Acta. 989:111-132 (1994)). Thepresent data now show that measuring changes in production of one ormore of these growth factors may predict when recovery has occurredafter previous cytotoxic therapy.

In sum, these data show that plasma FLT3-L levels predicted excessplatelet toxicity in 10 out of 12 patients (mean=225±106 pg/ml) and gavea false-positive in only 3 out of 27 other patients (mean of 80±41pg/ml). Plasma FLT3-L>135 pg/ml resulted in an 83% sensitivity and an85% and 89% specificity and accuracy, respectively, at predicting excesstoxicity from additional cytotoxic therapy. The positive likelihoodratio is 7.5 (95% confidence interval of 2.5-22.5) and the negativelikelihood ratio is 0.19 (95% confidence interval of 0.05-0.67).

Accordingly, elevated plasma FLT3-L in patients who received previouschemotherapy is a predictive measure of the stage of recovery of themarrow compartment. FLT3-L seems to identify the likelihood that thepatient will experience ≧grade 3 thrombocytopenia if additionalcytotoxic therapy is administered. Knowledge of marrow activity shouldpermit therapy that is more aggressive by establishing the earliestpossible time for dosing with any cytotoxic agent havingmyelosuppression as the dose-limiting toxicity.

Example 3 Adjustment of Red Marrow Dose by FLT3-L Ratio

Red marrow radiation doses were determined for 30 patients (20 males and10 females, all without bone marrow or bone involvement, 18 had priorchemotherapy) after receiving ¹³¹I-RAIT (activity range 2.1-8.9 GBq).Radiation dose estimates were calculated using two different methods ofred marrow cumulated activity and red marrow-to-blood activityconcentration ratio determinations for two dosimetric models, using bothmale and female and male-only masses and S values. Highest platelettoxicity grade at nadir (PTG), percent platelet count decrease (PPD),and platelet nadir counts (PN) were measured. FLT3-L levels (pg/ml) weredetermined by immunoassay prior to treatment; a normal FLT3-L level wasassumed to be 80 pg/ml. The red marrow radiation doses (cGy) wereadjusted for the patient's FLT3-L level when the patient's cytokinelevel exceeded the normal value. Marrow doses and FLT3-L adjusted marrowdoses were correlated to PTG, PPD, PN, and 1/PN. Administered activity,administered activity per unit body weight, and total body radiationdose were also correlated to these hematologic toxicity measures.

All the red marrow dose calculation schemes resulted in essentially thesame correlations with the hematologic toxicity measures. Poorcorrelations were observed between administered activity, administeredactivity per unit body weight, total-body radiation dose, or red marrowradiation dose and PTG, PPD, PN and 1/PN. All correlations improvedgreatly when the various predictors of toxicity were adjusted for thepatient's FLT3-L level. The highest correlation observed was between redmarrow dose or total body dose and 1/PN (r=0.86). Using an unadjustedred marrow dose to predict toxicity ≧Grade 3, there were 8 truepositive, but 13 false positive cases with 9 true negatives. However,using a FLT3-L-adjusted red marrow dose, there were 8 true positives,but only 2 false positives and 20 true negatives.

FLT3-L adjusted red marrow radiation doses provide improved correlationwith hematologic toxicity. Thus, elevated FLT3-L plasma levels prior toRAIT indicate increased radiosensitivity of the bone marrow and providebetter prediction of toxicity than red marrow radiation dose alone,leading to better treatment planning and minimization of toxicity byadjustment of administered activity.

Materials and Methods

Patients and Antibodies

Thirty patients (20 male and 10 female) enrolled in institutional reviewboard-approved Garden State Cancer Center clinical radioimmunotherapy(RAIT) trials were included in this study. No patient had known bonemarrow or bone metastases. The patients received RAIT with ¹³¹I labeledanti-carcinoembryonic antigen (CEA) NP4 (IgG or F(ab′)₂) or anti-CEAMN-14 (IgG or F(ab)₂, supplied by Immunomedics, Inc., Morris Plains,N.J.) (Juweid M E, Zhang C, Blumenthal R D, Hajjar G, Sharkey R M,Goldenberg D M). Prediction of hematologic toxicity afterradioimmunotherapy with ¹³¹I-labeled anticarcinoembryonic antigenmonoclonal antibodies. J Nucl Med. 1999; 40:1609-1616) with administeredactivities ranging from 2.1-8.9 GBq for the treatment of CEA-producingcancers. The treatment activity prescription was either a fixed activityof 2.8 GBq, an activity based on the patient's body surface area, or anactivity determined by a pretherapy tracer study performed 1-2 wk beforetreatment to deliver a prescribed red marrow radiation dose. Allinfusions were given intravenously over a 15-30 min time period, and allpatients were premedicated with Lugol's or supersaturated potassiumiodine solution and potassium perchlorate to decrease thyroid andgastric uptake of radioiodine. These labeled monoclonal antibodies areknown to not bind to crossreactive antigens, especially in the redmarrow (Sharkey R M, Goldenberg D M, Goldenberg H, et al. Murinemonoclonal antibodies against carcinoembryonic antigen: immunological,pharmacokinetic and targeting properties in humans. Cancer Res. 1990;50:2823-2831, Hansen H J, Goldenberg D M, Newman E, Grebenau R, SharkeyR M. Characterization of second generation monoclonal antibodies againstcarcinoembryonic antigen. Cancer. 1993; 71:3478-3485, Sharkey R M,Goldenberg D M, Murthy S, et al. Clinical evaluation of tumor targetingwith a high affinity anticarcinoembryonic-antigen-specific, murinemonoclonal antibody, MN-14. Cancer. 1993; 71:2081-2096). Twelve patientswere chemotherapy-naive and the remainder had multiple cycles ofprevious chemotherapy using various drugs and different duration rangingfrom 1 to 24 months since their previous treatment.

Blood and Total-Body Pharmacokinetics

Blood-cumulated activity concentrations were determined by countingsamples of whole blood in a calibrated gamma well counter to obtainblood activity concentrations at various time points after the end ofthe antibody activity infusion. Three to eight blood samples werecollected over the first 24 h, and then daily sampling was performedover the next 2-7 d. These time-activity concentration curves wereanalyzed using a nonlinear least squares curve fitting algorithm todetermine the slopes of the distribution (α) and elimination (β) phasesand their respective intercepts (A and B). These curves, which wereeither monophasic or biphasic, were then integrated to obtain the bloodcumulated activity concentration. Total body cumulated activities weredetermined using either whole-body gamma camera counts or hand-heldradiation probe counts obtained at multiple time pointspost-administration.

Plasma Cytokine Immunoassays

Blood samples were collected in all patients on the day of RAIT. Inaddition, blood samples were collected from five normal volunteers.Plasma FLT3-L in these blood samples was measured by a quantitativesandwich enzyme immunoassay using R&D Quantikine Immunoassay kits(Minneapolis, Minn.). Samples were run in duplicate and results wereread from a linear standard curve. The assay kits were sensitive (7pg/ml), specific, and showed no significant cross-reactivity with anyother murine or human cytokine. The purpose of the volunteer samplingwas to determine a normal FLT3-L level.

Toxicity Assessment

Myelotoxicity was graded according to the Radiation Therapy OncologyGroup (RTOG) criteria. All patients given therapeutic administrations of¹³¹I monoclonal antibodies were followed for hematologic toxicity bymonitoring complete peripheral blood cell counts weekly. Patient bloodwas collected prior to RAIT to establish the baseline peripheral whiteblood cell (WBC) and platelet (PLT) counts. When Grade 2 or higherthrombocytopenia or leukopenia developed, measurements were taken morefrequently until the nadir had been determined. The patients' bloodcounts were followed until complete hematologic recovery wasestablished. Since thrombocytopenia is often the dose-limiting factorfor RAIT, platelet toxicity grade (PTG), percent platelet decrease(PPD), and platelet nadir (PN) were used as the measures of toxicity inthis study. In addition, 1/PN was determined.

Red Marrow Dosimetry

Red marrow radiation dose was estimated in all patients based on themeasured cumulated activity in the whole blood and the measuredcumulated activity in the total body. The relative contribution of eachof these two components to the red marrow dose estimate is dependentupon the total body-to-blood cumulated activity ratio (Siegel J A,Stabin M G, and Sparks R B. Total body and red marrow dose estimates. JNucl Med. 2003; 44:320-321). Additional distinguishable source organcontributions could also be included (Siegel J A, Wessels B W, Watson EE, et al. Bone marrow dosimetry and toxicity for radioimmunotherapy.Antibody Immunoconj Radiopharm. 1990; 3:213-233); however, theirexpected contribution to red marrow dose has been estimated to be on theorder of 5% or less. A two-component equation (Bigler R E, Zanzonico PB, Leonard R, et al. Bone marrow dosimetry for monoclonal antibodytherapy. In: Schlaflke-Stelson A T, Watson E E, eds. FourthInternational Radiopharmaceutical Dosimetry Symposium. Oak Ridge: OakRidge Associated Universities; 1986: 535-544) was therefore used todetermine red marrow absorbed dose, since these patients do not havedisease in bone marrow or bone and the radioimmunotherapeutic agentsthey received do not bind to any blood, marrow, or bone elements. Thefirst component reflects the red marrow dose contribution associatedwith the activity distributed within the extracellular fluid space ofthe red marrow due to the circulating blood activity, and the secondcomponent reflects the absorbed dose contribution associated with theactivity in the remainder of the body, according to:D _(RM) =Ã _(RM) S(RM←RM)+Ã _(RB) ×S(RM←RB)  Eq. 1

where D_(RM) is the red marrow dose estimate, Ã_(RM) is the red marrowcumulated activity, Ã_(RB) is the remainder of the body cumulatedactivity obtained by subtracting the red marrow value, Ã_(RM), from thetotal body value, Ã_(TB), S(RM←RM) is the red marrow-to-red marrow Svalue, and S(RM←RB) is the remainder of the body-to-red marrow S value.Most investigators have used one of two dosimetric models, namely MIRD11 or MIRDOSE 3 (Stabin MG. MIRDOSE: personal computer software for usein internal dose assessment in nuclear medicine. J Nucl Med. 1996;37:538-546), for the needed S values in Equation 1. Therefore, both setsof S values and their associated masses were used to compare the redmarrow dose results. Further, MIRDOSE 3 explicitly provides S values forfemales; therefore, an additional red marrow dose comparison wasperformed using both male and female versus male-only masses and Svalues. The model masses were always adjusted for patient weight throughmultiplication by the total body mass of the patient divided by thetotal body mass of the model; all S values were adjusted using theinverse of this mass relationship (linear mass-based scaling of the Svalues for ¹³¹I is not strictly correct since the photon absorbedfractions do not scale linearly with weight; however, this approximationgives adequate results). The red marrow mass of the adult male model is1.5 kg and 1.12 kg for MIRD 11 and MIRDOSE 3, respectively, and thetotal-body mass of the adult male model is 69.88 kg and 73.7 kg for MIRD11 and MIRDOSE 3, respectively. For females, the MIRDOSE 3 red marrowmodel mass is 1.05 kg and the model total-body mass is 58 kg. It shouldbe noted that the remainder of the body-to-red marrow S value wasdetermined not only by using the patient-specific approach of adjustingthe model S values and masses by the patient total body weight, butalso, since there is no bone activity uptake in the patients studied,the bone component (i.e., trabecular and cortical) contribution to thisterm was explicitly subtracted (Stabin M G, Siegel J A, Sparks R B,Eckerman K F, Breitz H B. Contribution to red marrow absorbed dose fromtotal body activity: a correction to the MIRD method. J Nucl Med. 2001;42:492-498).

The red marrow cumulated activity, Ã_(RM), in Equation 1 was determinedusing two approaches: $\begin{matrix}{{\overset{\sim}{A}}_{RM} = {\left\lbrack \overset{\sim}{A} \right\rbrack_{blood}m_{{RM},{model}}\frac{m_{{TB},{patient}}}{m_{{TB},{model}}}{CF}}} & {{Eq}.\quad 2} \\{{\overset{\sim}{A}}_{RM} = {1.443\quad T_{e,{blood}}\frac{m_{RM}}{m_{blood}}{CF}}} & {{Eq}.\quad 3}\end{matrix}$

where [Ã]_(blood) is the blood cumulated activity concentration obtainedfrom analysis of the blood activity concentration-time curve,m_(RM,model) is the red marrow mass of the respective dosimetric model,m_(TB,patient) is the total body mass of the patient, m_(TB,model) isthe total body mass of the respective model, T_(e,blood) is the bloodeffective half-time obtained from analysis of the blood activityconcentration-time curve (if the blood activity concentration-time curvewas biphasic, T_(e,blood) is replaced by Σ_(i)f_(i)(T_(i))_(e,blood),where f_(i) is the activity concentration fraction of the i-thexponential component and (T_(i))_(e,blood) is the effective half-timeof the i-th exponential component). Since the red marrow and bloodmasses are assumed to vary similarly as a function of patient weight,the value of the mass ratio in Equation 3 is assumed to be a fixedvalue. Finally, CF is a correction factor for the marrow-to-bloodactivity concentration ratio. Originally, the correction factor, CF, wasset at unity (1), but other investigators have shown this value to betoo conservative. CF is currently assigned either a fixed value ofbetween 0.2-0.4 or a value of 0.19/(1-hematocrit) (Sgouros G. Bonemarrow dosimetry for radioimmunotherapy: theoretical considerations. JNucl Med. 1993; 34:689-694, Siegel J A, Lee R A, Pawlyk D A, Horowitz JA, Sharkey R M, Goldenberg D M. Sacral scintigraphy for bone marrowdosimetry in radioimmunotherapy. Nuci Med Biol. 1989; 16:553-559, SiegelJ A, Pawlyk D A, Lee R A, et al. Tumor, red marrow, and organ dosimetryfor ¹³¹I-labeled anti-carcinoembryonic antigen monoclonal antibody.Cancer Res. 1990; 50 (suppl):1039s-1042s, Siegel J A, Lee R A, HorowitzJ A, et al. Bone marrow dosimetry: marrow-to-blood activityconcentration ratio [abstract]. J Nucl Med. 1990; 31:788). For thisanalysis, two approaches have been used for red marrow radiation dosecomparison: a fixed CF of 0.3 and a CF determined using the value of0.19/(1-hematocrit).

All red marrow radiation doses (cGy) were adjusted for the patient'sFLT3-L level when the patient's cytokine levels exceeded normal values.Marrow doses and FLT3-L adjusted marrow doses were correlated to PTG,PPD, PN and 1/PN. In addition, administered activity, administeredactivity per unit body weight, and total body dose (equal to Ã_(TB)multiplied by the mass-adjusted total body-to-total body S value) werealso correlated to these measures of hematologic toxicity.

Results

The FLT3-L level was determined in the volunteers to be 52±14 pg/ml;therefore a normal value of FLT3-L was assumed to be 80 pg/ml(mean±2SD). The red marrow radiation doses (cGy) were adjusted for thepatient's FLT3-L level (FLT3-L level/80) when the patient's cytokinelevel exceeded 80 pg/ml. All the red marrow dose calculation schemesresulted in essentially the same correlations with the various measuresof hematologic toxicity (Table 5):

Use of MIRD 11 and MIRDOSE 3 S values and masses yielded similarcorrelations.

Use of male-only parameters resulted in similar correlations to use ofboth male and female model parameters.

Use of the two methods for red marrow cumulated activity determination(Equations 2 and 3) resulted in similar-dose-toxicity correlations.

Use of constant red marrow-to-blood activity concentration ratio(CF=0.3) yielded similar results to use of the more patient-specific CFdetermination.

Use of 1/platelet nadir yielded better correlations than use of plateletgrade, percent decrease in platelets, or platelet nadir. TABLE 5Correlation Coefficients Correlation coefficients (r) Red Marrow Dose(cGy) Total Body Activity Activity/Body CF = 0.3 CF = 0.19/(1-hct) Dose(cGy) (GBq) Weight (GBq/k

1. MIRDOSE 3 A. All Males i. Equation 3 PTG 0.28 (0.70) 0.25 (0.70) 0.23(0.68) 0.05 (0.61) 0.28 (0.72) PPD 0.15 (0.48) 0.10 (0.47) 0.06 (0.46)0.15 (0.51) 0.33 (0.59) PN 0.22 (0.76) 0.20 (0.76) 0.16 (0.75) 0.03(0.60) 0.18 (0.75) 1/PN 0.20 (0.86) 0.19 (0.85) 0.17 (0.86) 0.21 (0.53)0.04 (0.79) ii. Equation 2 PTG 0.31 (0.68) 0.28 (0.68) PPD 0.15 (0.46)0.12 (0.46) PN 0.27 (0.75) 0.24 (0.74) 1/PN 0.21 (0.84) 0.17 (0.82) B.Males & Females Equation 3 PTG 0.31 (0.68) 0.27 (0.68) PPD 0.15 (0.46)0.11 (0.45) PN 0.27 (0.74) 0.24 (0.74) 1/PN 0.20 (0.86) 0.18 (0.85) 2.MIRD 11 Equation 3 PTG 0.31 (0.67) 0.26 (0.67) PPD 0.17 (0.46) 0.12(0.46) PN 0.28 (0.73) 0.23 (0.71) 1/PN 0.20 (0.85) 0.16 (0.82)

All predictors of toxicity (administered activity, administered activityper unit body weight, total body dose, and red marrow dose) whenadjusted for the patient's observed FLT3-L level yielded strongercorrelations than when non-adjusted.

Adjusted red marrow and total-body dose yielded better correlations thanadjusted administered activity (GBq) or adjusted administered activityper unit body weight (GBq/kg) when using 1/PN as the measure ofhematologic toxicity.

The correlation coefficients for PN versus radiation dose weredetermined using an exponential function; all other correlations weredetermined using linear regression. The comparisons of PN with theFLT3-L adjusted predictors of toxicity are shown in FIG. 4, and thecomparisons of 1/PN with the FLT3-L adjusted toxicity predictors areshown in FIG. 5.

Poor correlations were observed between the administered activity andPTG, PPD, PN and 1/PN (r=0.05, 0.15, 0.03 and 0.21, respectively) andthe administered activity per unit body weight and these hematologictoxicity measures (r=0.28, 0.33, 0.18 and 0.04, respectively). Similarpoor correlations were observed between red marrow radiation dose andPTG, PPD, PN and 1/PN (r=0.28, 0.15, 0.22, and 0.20, respectively).Correlations between FLT3-L-adjusted marrow dose and PTG, PPD, PN and1/PN were greatly improved (r=0.70, 0.48, 0.76, and 0.86, respectively),as were the correlations for administered activity and administeredactivity per unit body weight. Correlations between FLT3-L-adjustedtotal body dose, using only the MIRDOSE 3 dosimetric model, and PTG,PPD, PN and 1/PN were 0.68, 0.46, 0.75, and 0.86, respectively.

Only 8 patients had a PTG of 3 or 4. RM dose adjusted for FLT3-L versus1/PN for these patients resulted in a correlation coefficient of 0.85.FLT3-L-adjusted TB dose, administered activity and administered activityper unit body weight versus 1/PN resulted in correlation coefficients of0.81, 0.14, and 0.60, respectively. The other 22 patients had grade 0-2platelet toxicities; adjusted RM dose versus 1/PN for these patientsresulted in a correlation coefficient of 0.18. Adjusted TB dose,administered activity and administered activity per unit body weightversus 1/PN resulted in correlation coefficients of 0.33, 0.42, and0.27, respectively.

The FLT3-L-adjusted red marrow doses, using the male MIRDOSE 3 model,Equation 3 and a CF of 0.3, were compared to the FLT3-L-adjustedadministered activities per unit body weights and unadjusted red marrowdose as predictors of Grade 3 or higher toxicity by determiningsensitivity, specificity, accuracy, positive predictive value (PPV) andnegative predictive value (NPV). Using a threshold value of 200 cGy forthe adjusted red marrow dose, there were 8 true positives, 0 falsenegatives, 2 false positives and 20 true negatives, resulting in asensitivity, specificity, accuracy, PPV, and NPV of 100%, 90.9%, 93.3%,80%, and 100%, respectively. Although the number of patients with Grade3 or higher hematologic toxicity was low (n=8), there were 22 patientswho did not develop Grade 3 or higher toxicity, and in these patientsthere were no false negatives but 20 true negatives. Using a thresholdvalue of 100 cGy for red marrow dose by itself, there were 8 truepositives, 0 false negatives, 13 false positives and 9 true negatives,resulting in a sensitivity, specificity, accuracy, PPV, and NPV of 100%,40.9%, 56.7%, 38.1%, and 100%, respectively. Using a threshold value of74 MBq/kg for the adjusted activity per body weight, there were 8 truepositives, 0 false negatives, 6 false positives, and 16 true negativesresulting, in a sensitivity, specificity, accuracy, PPV, and NPV of100%, 72.7%, 80%, 57.1%, and 100%, respectively.

Correlations for dose-toxicity were as high as 0.86 betweenFLT3-L-adjusted radiation dose and the inverse of platelet nadir as themeasure of hematologic toxicity. Correlations with this latter parameterwere much higher compared to all other measures of hematologic toxicity(platelet toxicity grade, percent platelet decrease, and plateletnadir); the correlation coefficients jumped from a range ofapproximately 0.5-0.8 up to almost 0.9. The use of 1/PN appears totransform the PN-hematologic toxicity predictor curves to theanticipated shape of a dose-response curve (i.e., at low dose limitedtoxicity is observed followed by increasing toxicity at higher dose in anonlinear fashion). The classic sigmoidal curve was not observed,presumably due to the fact that the calculated dose levels were not highenough to establish this shape. Thus, it is reasonable to expect thatlinear correlation with 1/PN versus the various predictors of toxicitywould result in a much stronger correlation than the other toxicitymeasures.

In this limited patient population, clear distinctions were not found inthe correlations between patients with long or short effectivehalf-times in blood. This explains why the FLT3-L adjustment oftotal-body dose and administered activity per unit body weightcorrelated with observed toxicity as well as red marrow absorbed dose(the correlations involving administered activity were not as good).When patients were separated in terms of the severity of their bonemarrow toxicity (i.e., Grade 3 or 4 platelet toxicity versus thosepatients with a PTG of 0-2), both red marrow and total-body doseresulted in stronger correlations than administered activity andadministered activity per unit body weight. In addition, FLT3-L adjustedred marrow dose resulted in higher specificity, accuracy, and positivepredictive value compared to adjusted activity per unit body weight andred marrow dose by itself. Furthermore, when using FLT3-L-adjusted redmarrow dose as a predictor of hematologic toxicity, there were no falsenegatives and 20 of the 22 patients with less than Grade 3 toxicity weretrue negatives.

The blood-based red marrow dosimetry approaches in this study arejustifiable since no patient had bone marrow and/or bone metastases andthe radiolabeled monoclonal antibodies administered do not bind to anyblood, marrow, or bone components, with one caveat. Patients who arerecovering from chemotherapy may have hyperproliferating bone marrowwith enhanced radioantibody uptake (Juweid M, Sharkey R M, Siegel J A,Behr T, Goldenberg D M. Estimates of red marrow dose by sacralscintigraphy in radioimmunotherapy patients having non-Hodgkin'slymphoma and diffuse bone marrow uptake. Cancer Res. 1995; 55(suppl):5827s-5831 s). If such involvement were present, red marrowdosimetry would need to take this into consideration. In such patientsimage-based red marrow dose estimates have been shown to better predictmyelotoxicity (Juweid M, Sharkey R M, Siegel J A, Behr T, Goldenberg DM. Estimates of red marrow dose by sacral scintigraphy inradioimmunotherapy patients having non-Hodgkin's lymphoma and diffusebone marrow uptake. Cancer Res. 1995; 55 (suppl):5827s-5831s, Macey D J,DeNardo S J, DeNardo G L. Estimation of radiation absorbed doses to redmarrow in radioimmunotherapy. Clin Nucl Med. 1995; 20:117-125). It hasalso recently been claimed that image-based red marrow dose estimatesmight improve the prediction of toxicity for non-marrow targeting ⁹⁰Yantibody therapy (Shen S, Meredith R F, Duan J, Brezovich I A, Robert F,Lobuglio A F. Improved prediction of myelotoxicity using imaging doseestimate for non-marrow targeting ⁹⁰Y-antibody therapy [abstract]. JNucl Med. 2001; 5 (suppl): 22P).

CONCLUSION

FLT3-L adjusted red marrow and total-body radiation doses provideimproved correlation with hematologic toxicity. The adjusted absorbeddoses provided a stronger dose-toxicity correlation than the use ofsimpler empirical parameters, such as administered activity andadministered activity per unit body weight. Elevated FLT3-L plasmalevels prior to RAIT indicate increased radiosensitivity of the bonemarrow, and use of this measurement to adjust calculated red marrow ortotal body radiation doses provides a significantly better prediction oftoxicity than radiation dose alone, leading to better treatment planningand minimization of toxicity by optimization of administered activity.Improved methods for red marrow absorbed dose estimates will allow foreven better treatment optimization. Further, in those patientsidentified to be at low risk for toxicity, the administered activity maybe increased, potentially leading to a greater treatment benefit.

The foregoing detailed description and Examples are merely meant to beillustrative, and not limiting in any way. The artisan will immediatelyappreciate that there are other aspects falling within the inventionthat are not specifically exemplified. All references cited above areherein incorporated in their entirety to the same extent as if each wasindividually incorporated.

1. A method of determining the dose of a hematopoietic cytokine to beadministered to a patient before or after a myelosuppressive agent is tobe administered to the patient, comprising: (a) measuring the level ofat least one cytokine in a sample from the patient; (b) comparing thelevel of the at least one cytokine to the levels of the cytokine innormal subjects; (c) determining the dose of the hematopoietic cytokineto be administered to the patient; and (d) administering thehematopoietic cytokine to the patient.
 2. The method of claim 1, whereinthe levels of two or more cytokines are measured.
 3. The method of claim1, wherein the at least one measured cytokine and the hematopoieticcytokine to be administered to the patient are the same.
 4. The methodof claim 1, wherein the at least one measured cytokine and thehematopoietic cytokine to be administered to the patient are different.5. The method of claim 1, wherein the level of the at least one cytokineis measured in the patient before administering the myelosuppressiveagent to the patient.
 6. The method of claim 1, wherein the level of theat least one cytokine is measured in the patient after administering themyelosuppressive agent to the patient.
 7. The method of claim 1, whereinthe level of the at least one cytokine is measured in the patient beforeand after administering the myelosuppressive agent to the patient. 8.The method of claim 1, wherein the at least one cytokine to be measuredcomprises G-CSF.
 9. The method of claim 1, wherein the at least onecytokine to be measured comprises GM-CSF.
 10. The method of claim 1,wherein the at least one cytokine to be measured comprises G-CSF andGM-CSF.